Adjuvant compositions and methods for enhancing immune responses to polynucleotide-based vaccines

ABSTRACT

The invention provides adjuvants, immunogenic compositions, and methods useful for polynucleotide-based vaccination and immune response. In particular, the invention provides an adjuvant of cytofectin:co-lipid mixture wherein cytofectin is GAP-DMORIE.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. application Ser.No. 13/031,064, filed Feb. 18, 2011, which is a divisional applicationof U.S. application Ser. No. 11/889,980, filed Aug. 17, 2007, nowabandoned, which is a continuation application of U.S. application Ser.No. 10/391,216, filed Mar. 19, 2003 (now U.S. Pat. No. 7,582,613, issuedon Sep. 1, 2009), which is a divisional application of U.S. applicationSer. No. 09/534,943, filed Mar. 24, 2000 (now U.S. Pat. No. 6,586,409,issued on Jul. 1, 2003), which claims the benefit of U.S. ProvisionalApplication No. 60/126,340, filed Mar. 26, 1999; said U.S. applicationSer. No. 10/391,216 is also a continuation of U.S. application Ser. No.09/937,604, filed Sep. 26, 2001 (now U.S. Pat. No. 7,105,574, issued onSep. 12, 2006), which is a 35 U.S.C. §371 National Phase filing ofInternational Application No. PCT/US00/08282, with an internationalfiling date of Mar. 24, 2000, which claims the benefit of said U.S.Provisional Application No. 60/126,340; said U.S. application Ser. No.09/937,604 is also a continuation of said U.S. application Ser. No.09/534,943, which claims the benefit of said U.S. ProvisionalApplication No. 60/126,340; each of the above application sisincorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to adjuvants, immunogeniccompositions, and methods useful for polynucleotide-based vaccination.The present invention provides compositions and methods useful forenhancing immune response, especially the humoral immune response ofvertebrates to polynucleotide-based vaccines. In particular, the presentinvention provides an adjuvant of cytofectin:co-lipid mixture whereinthe cytofectin is(±)-N-(3-aminopropyl)-N,N-dimethyl-2,3-bis(syn-9-tetradeceneyloxy)-1-propanaminiumbromide (GAP-DMORIE).

In the late 1980s, it was discovered that direct intramuscular (i.m.)injection of lipid-DNA complexes results in measurable proteinexpression, and that “naked” plasmid DNA (pDNA) is taken up andexpressed in muscle to a greater extent than lipid-DNA complexes(Felgner, Scientific American, 276(6), 102-106 (1997)).

One of the first applications of pDNA injection technology was theinduction of an immune response. In 1991, it was first reported thatmice could be immunized against HIV gp120 by i.m. vaccination with gp120plasmid DNA (Felgner et al., Nature, 349, 351-352 (1991)), and that micecould be protected from a lethal challenge of influenza virus after DNAimmunization with influenza nucleoprotein (NP) antigen. Protectionobtained after immunization with the highly conserved NP antigenextended across 2 different viral strains (Ulmer et al., CurrentOpinions In Immunology, 8, 531-536 (1996)). Numerous publications in thefield of polynucleotide-based vaccination followed thereafter (e.g.,Boyer et al., J. Med. Primatology, 25(3), 242-250 (1996); Boyer et al.,Nature Medicine, 3(5), 526-532 (1997); Davis et al., Vaccine, 15(8),849-852 (1997); Wang et al., Vaccine, 15(8), 821-825 (1997); Agadjanyanet al., Current Topics In Microbiology And Immunology, 226, 175-192(1998); Heppell et al., Fish & Shellfish Immunology, 8(4), 271-286(1998); Lodmell et al., Nature Medicine, 4(8), 949-952 (1998);Vanderzanden et al., Virology, 246(1), 134-144 (1998)).

A major problem frequently encountered in the course ofpolynueleotide-based vaccination is insufficient or suboptimal humoralresponse. Often, the antigens or immunogens encoded by thepolynucleotide are expressed in vivo, but they are not sufficientlyimmunogenic to raise the antibody titer in the organism to sufficientlevels to provide protection against subsequent challenge and/or tomaintain the potential for generating therapeutically active antibodylevels over extended time periods. To obtain a stronger humoral and/orcellular response, it is common to administer such vaccines in animmunogenic composition containing an adjuvant, a material whichenhances the immune response of the patient to the vaccine. Adjuvantsare useful generally for improving the immune response of an organism toa particular immunogen and are commonly included in vaccine compositionsto increase the amount of antibodies produced and/or to reduce thequantity of immunogen and the frequency of administration.

A variety of adjuvants have been reported to effect differing levels ofimmune response enhancement to polynucleotide-based vaccination.Examples of such adjuvant materials include semi-synthetic bacterialcell wall-derived mono-phosphoryl lipid A (Sasaki, S., et al., Infectionand Immunity 65(9), 3250-3258 (1997)), small molecule immunostimulators(Sasaki, S., et al., Clin. Exp. Immunol. 111, 30-35 (1998)), andsaponins (Sasaki, S., et al., J. Virology 72(6), 4391-4939 (1998)). Theimmune response from i.m. pDNA vaccination has also been enhancedthrough the use of cationic lipids (Ishii, N., et al., Aids Res. Hum.Retroviruses 13(16), 1421-1428 (1997)), Okada, E., et al., J. Immunology159, 3638-3647 (1997); Yokoyama, M., et al., FEMS Immunol. Med.Microbiol. 14, 221-230 (1996); Gregoriadis, G., et al., FEBS Letters402, 107-110 (1997); Gramzinski, R. A., et al., Molecular Medicine 4,109-118 (1998); Klavinskis, L. S., et al., Vaccine 15(8), 818-820(1997); Klavinskis, L. S., et al., J. Immunology 162, 254-262 (1999);Etchart, N., et al., J. Gen. Virology 78, 1577-1580 (1997); Norman, J.,et al., in Methods in Molecular Medicine, Vol. 9; DNA Vaccines: Methodsand Protocols, D. B. Lowrie and R. Whalen, eds., Chapter 16, pp. 185-196(1999)). Cationic lipids were originally studied as cytofectins toenhance delivery of pDNA into cells in vitro; however, furtherdevelopment has led to successful specific applications of proteindelivery in vivo (Wheeler, C. J., et al., Proc. Natl. Acad. Sci. USA 93,11454-11459 (1996); Stephan, D. J., et al., Human Gene Therapy 7,1803-1812 (1996); DeBruyne, L. A., et al., Gene Therapy 5, 1079-1087(1998)). Accordingly, such cytofectins may be useful for vaccineapplications by enhancing delivery of the pDNA into the cellsresponsible for giving rise to the humoral arm of the immune response,thereby increasing antibody titer levels.

Commonly used adjuvants show low levels of immune response enhancementfor vaccination (typically less than 3-fold) and possess undesirabletoxicological and manufacturing profiles. In addition, cationic lipidsused previously for vaccination show only low levels of humoralenhancement. There is a need for more adjuvant compositions useful forenhancing the immune response of vertebrates to immunization, especiallyto pDNA vaccination.

BRIEF SUMMARY OF THE INVENTION

The present invention is directed to adjuvant and immunogeniccompositions and to methods for the polynucleotide-based vaccination ofa vertebrate, to help protect the vertebrate from a disease, to treat adiseased vertebrate, or both. In certain preferred embodiments, thepresent invention is directed to a method for immunizing a vertebrate byadministering to the vertebrate a composition comprising apolynucleotide that encodes for an immunogen, wherein the polynucleotideis complexed with an adjuvant composition comprising GAP-DMORIE.Preferably, the composition may comprise one or more co-lipids. Theimmunogen-encoding polynucleotide, upon incorporation into the cells ofthe vertebrate, produces an immunologically effective amount of animmunogen (e.g., an immunogenic protein). The adjuvant composition ofthe present invention enhances the immune response of the vertebrate tothe immunogen,

One aspect of the present invention is an adjuvant compositioncomprising a mixture of one or more cytofectins and one or moreco-lipids, which adjuvant composition is useful for enhancing thehumoral immune response of a vertebrate to an immunogen. Preferably, theadjuvant composition includes the cytofectin GAP-DMORIE and one or moreco-lipids. Preferably also, the co-lipid is a neutral lipid such as, forexample, a phosphatidylethanolamine. More preferably, the co-lipid is1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE),1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (DPyPE), and/or1,2-dimyristoyl-glycer-3-phosphoethanolamine (DMPE). Most preferably,the co-lipid is DPyPE.

Another aspect of the present invention is an immunogenic compositioncomprising one or more immunogens and an adjuvant composition comprisingthe cytofectin GAP-DMORIE and one or more co-lipids. In certainembodiments, the source of the immunogen is an immunogen-encodingpolynucleotide, such as in the case of a pDNA vaccine. Preferably, inthose embodiments, the pDNA or polynucleotide is complexed with anadjuvant composition comprising GAP-DMORIE and one or more co-lipids.

Another aspect of the present invention is a method for immunizing avertebrate by administering to the vertebrate an immunogenic compositioncomprising a complex of one or more immunogen-encoding polynucleotidesand GAP-DMORIE in an amount sufficient to generate an immune response tothe encoded immunogen. Preferably, the immunogenic composition furtherincludes one or more co-lipids such as, for example, DOPE and/or DPyPE.Most preferably, the co-lipid is DPyPE.

The present invention, in contrast to the prior art, is useful forenhancing the humoral immune response of a vertebrate to apolynucleotide-based vaccine, through the use of GAP-DMORIE. Elevationof antibody levels is particularly advantageous in applications whereantibody levels from the immunogen-encoding polynucleotide alone aresub-optimal. In a related advantage, if the desired level of antibodiesis produced with a given dose of pDNA, the amount of pDNA necessary toreach the predetermined antibody titer level can be reached using alower pDNA dose. For pDNA vaccination applications, this advantage isimportant because acceptable vaccination volumes, coupled withfunctional limits on the concentration of pDNA, define an upper limit ona given vaccine dose. This advantage is particularly beneficial forvaccines containing multiple plasmids, each of which must be present insufficient quantity to elicit an immune response to its particulartransgene.

BRIEF DESCRIPTION OF THE FIGURES

The foregoing aspects and advantages of the present invention will bereadily apparent to one skilled in the art upon reference to the figuresand the detailed description which follows.

FIG. 1 illustrates the diagrams of Plasmid DNAs. Each vector has a pUC19origin of replication and a kanamycin resistance gene for plasmid growthin E. coli bacteria. CMV=human cytomegalovirus promoter and enhancer;CMV−A=human cytomegalovirus intron A; mRGB=modified rabbit β-globinpolyadenylation signal; BGH=bovine growth hormone polyadenylationsignal.

FIG. 2 illustrates the chemical structures for the cytofectin GAP-DMORIEand the co-lipids DOPE and DPyPE, along with structurally relatedcytofectins.

FIG. 3 is a bar graph demonstrating that the structural elements ofcytofectins determine the level of antibody stimulation uponadministration. Mice were immunized using pDNA coding for influenzanuclear protein (NP), complexed with various cytofectins (identified onthe horizontal axis) formulated as a 1:1 (mol:mol) mixture with DOPEco-lipid. Each animal in the test group (five animals per group) wasinjected at day “0” and at 3 weeks (boost injection) with 5 μg pDNA in50 μl physiological saline per leg in the rectus femoris muscle, eitheralone or as a complex with a cytofectin:co-lipid adjuvant. After 6 weeks(3 weeks after the boost), serum was removed from the animals and the NPantibody titers were determined by serial dilution using an ELISA assay.Cytofectin:co-lipid enhancement was evaluated using the ratio of (i) thegeometric mean titer (GMT) from a cytofectin-augmented transfectiongroup to (ii) the GMT from pDNA transfection alone, using an equivalentcontrol group of animals.

FIG. 4 is a bar graph illustrating the differential enhancement ofanti-NP antibody responses to cytofectins by using DPyPE instead of DOPEas the co-lipid in the adjuvant composition. Mice were immunized andanalyzed as described above in connection with FIG. 2.

FIG. 5 illustrates pDNA-Vaxfectin vaccination dose response and timecourse. BALB/c mice (8-10 weeks old) received bilateral intramuscularinjections of 1 μg, 5 μg or 25 μg naked VR4700 plasmid DNA encodinginfluenza nuclear protein (NP) in 50 μl PBS per muscle (thus, 2 μg, 10μg or 50 μg total pDNA per time point). A second set of mice receivedthe same pDNA doses formulated with Vaxfectin using a constantpDNA:cationic lipid molar ratio of 4:1. Boost injections were given ondays 21 and 42 (arrows). Anti-NP titers were determined from serumsamples at 3, 6 and 9 weeks. The lines represent average anti-NPantibody titers+S.E.M. (n=5 mice per group).

FIGS. 6A and 6B illustrate Vaxfectin formulation optimization. Controlmice received bilateral intramuscular injections of 5 μg naked VR4700plasmid DNA encoding influenza nuclear protein (NP) in 50 μl PBS permuscle (white bars). The test groups received an equivalent pDNA doseformulated with Vaxfectin at the indicated pDNA:cationic lipid molarratios (black bars). Boost injections were identical to the initialinjections, and were given on day 21. Total NP-specific IgG antibodytiters were determined from serum samples on day 42 (3 weeks after theboost). The bars represent average anti-NP titers from two separateexperiments (n=5-15 mice per group).

FIGS. 7A and 7B illustrate the duration of elevated antibody titersinduced by Vaxfectin. Mice received bilateral intramuscular injectionsof either 5 μg naked VR4700 plasmid DNA encoding influenza nuclearprotein (NP) in 50 μl PBS per muscle, or the same amount of pDNAformulated with Vaxfectin at a pDNA:cationic lipid molar ratio of 4:1.Identical boost injections were given either on day 21 (A), or on day 21and again at 3 months (B) (arrows). Total NP-specific IgG antibodytiters were determined from serum samples at various time points. Thelines represent average anti-NP titers+S.E.M. (n=4-10 mice per timepoint).

FIGS. 8A, 8B, and 8C illustrate that pDNA formulated with Vaxfectininduces CTL responses that are as robust as those induced with nakedpDNA. (A) Mice received bilateral intramuscular injections of 5 μgVR4700 plasmid DNA encoding influenza nuclear protein (NP) in 50 μl PBSper muscle on day 0, 21, 42 and 63. A second set of mice received thesame pDNA dose formulated with Vaxfectin at the indicated pDNA:cationiclipid molar ratios. (B) Mice received bilateral intramuscular injectionsof 1 or 25 μg VR4700 plasmid in 50 μl PBS per muscle on day 0, 21, 42and 63. A second set of mice received the same pDNA doses formulatedwith Vaxfectin at a pDNA:cationic lipid molar ratio of 4:1. (C) Micereceived bilateral intramuscular injections of 5 μg VR4700 plasmid in 50μl 150 mM NaP per muscle on day 0 and 21. A second set of mice receivedthe same pDNA dose formulated with Vaxfectin at a pDNA:cationic lipidmolar ratio of 4:1. All CTL assays were performed 4-4.5 months after thefirst injection. The lines represent average specific lysis (n=4-5 miceper group).

FIG. 9 illustrates the effect of Vaxfectin on β-galactosidase (β-Gal)expression in muscle. Mice received intramuscular injections of 5 μgnaked VR1412 plasmid encoding β-galactosidase. A second group of micewas injected with 5 μg VR1412 formulated with Vaxfectin at apDNA:cationic lipid molar ratio of 4:1. At the indicated time points,quadriceps muscles were harvested and assayed for β-Gal activity. Thelines represent average reporter gene expression per muscle±S.E.M.(n=10-20 muscles per group).

FIG. 10 illustrates that Vaxfectin enhances humoral immune response inrabbits, Total IgG antibody titers in rabbit serum after i.m. injectionof VR4700 plasmid DNA encoding influenza nuclear protein (NP) are shown.New Zealand White rabbits (5-6 months old) received a single unilateralinjection of either 150μg VR4700 plasmid alone or formulated withVaxfectin (pDNA:cationic lipid=4:1 molar ratio) in 300 μl PBS. In onegroup of animals (triangles), both pDNA and pDNA-Vaxfectin were injectedusing needle and syringe. In another group of rabbits (circles), pDNAand pDNA-Vaxfectin were injected using a Biojector needle-free injectiondevice. On day 42 (arrow), rabbits were given an identical boostinjection in the contralateral quadriceps muscle. Anti-NP titers weredetermined from serum samples collected prior to immunization, and atweeks 3, 6, 7, 9, and 13. The lines represent average anti-NPtiters+S.E.M. (n=4 rabbits per group).

FIGS. 11A, 11B, 11C, 11D, and 11E illustrate that Vaxfectin enhancesantigen specific serum antibody responses to 5 different pDNA encodedmodel antigens. BALB/c mice were immunized with injections of 5 μgpDNA+/−Vaxfectin into each rectus femoris muscle at 0 and 3 weeks. Datashown are the mean antigen specific IgG titers (+/−SEM) for seracollected 1 day prior to the boost at 3 weeks and at 6 weeks. (n=20 forall groups, except for NP where n=29 for Naked NP pDNA; n=30 for NPpDNA/Vaxfectin and mouse Id where n=19 for naked pDNA.) A)Anti-influenza NP IgG titers; B) Anti-influenza HEL IgG titers; C)Anti-β-gal IgG titers; D) Anti-Mouse Id IgG titers; E) Anti-Factor IXIgGtiters. *Statistically significant difference from titers obtained withnaked pDNA, p≦0.05.

FIGS. 12A, 12B, 12C, and 12D illustrate that immunization with pDNAformulated with cytofectin induces antigen specific CTL lysis of targetcells coated with antigen derived peptides. BALB/c mice were immunizedwith injections of 5 μg pDNA+/−Vaxfectin into each rectus femoris muscleat 0 and 3 weeks. Spleens were harvested 11-12 weeks following theinitial immunizations and stimulated for 5-6 days with 1 μM NP₁₄₇₋₁₅₅ orβ-gal₈₇₆₋₈₈₄ peptide and 0.5 U/ml of recombinant murine IL-2. Datapresented are the average % lysis for 5 spleens in each group. Similarresults were obtained in a second assay for both NP and β-gal specificCTL. A) P815 target cells pulsed with NP₁₄₇₋₁₅₅ peptide; B) UnpulsedP815 target cells; C) P815 target cells pulsed with β-gal₈₇₆₋₈₈₄peptide; D) Unpulsed P815 target cells.

FIGS. 13A and 13B illustrate the Th1 type isotype profiles of antigenspecific antibodies induced with 5 different pDNA encoded modelantigens. Serum titers of antigen specific sub-isotypes are presented asa percentage of the sum of IgG1 and IgG2a titers. (n=20 for all groups,except for NP where n=29 for Naked NP pDNA; n=30 for NP pDNA/Vaxfectinand mouse Id where n=19 for naked pDNA.) A) Percent of IgG1 and IgG2a at6 weeks following naked pDNA immunizations. B) Percent of IgG1 and IgG2aat 6 weeks following pDNA/Vaxfectin immunizations.

FIGS. 14A and 14B illustrate Th1 type cytokine secretion profiles ofsplenocytes from pDNA/Vaxfectin immunized mice. Spleens were harvested11-12 weeks following the initial immunizations and were stimulated for72 hours with 5 μg/ml of purified NP or β-gal protein. IFN-γ and IL-4 inculture supernatants were determined by ELISA. The data presented arethe average concentration of cytokine from cultures of stimulatedsplenocytes less the concentration of cytokine from cultures ofunstimulated splenocytes (+/−SEM). A) Antigen specific IFN-γ response ofsplenocytes from naked pDNA and pDNA/cytofectin immunized mice (n=10 foreach group). B) Antigen specific IL-4 response of splenocytes from nakedpDNA and pDNA/Vaxfectin immunized mice (n=10 for each group).

DETAILED DESCRIPTION OF THE INVENTION

It will be apparent to one skilled in the art, in view of the followingdetailed description and the claims appended hereto, that varioussubstitutions and modifications may be made to the present inventionwithout departing from the scope of the invention as claimed.

The present invention is directed to the polynucleotide-basedimmunization of a vertebrate, to protect from or treat a vertebrate witha disease condition. The present invention includes the use ofcytofectin, especially GAP-DMORIE in adjuvants, immunogeniccompositions, and methods for immunizing a vertebrate, especially withpolynucleotude-based immunogen.

The adjuvant composition of the present invention includes one or morecytofectins and, in preferred embodiments, one or more co-lipids.Cytofectins are cationic lipids. In one embodiment, cytofectin isGAP-DMORIE, which has a structure corresponding to a2,3-dialkoxy-propanaminium skeleton possessing a unique combination oftwo linear fourteen-carbon mono-unsaturated alkyl chains and apropylamine substituent on the quaternary nitrogen (See FIG. 2).

GAP-DMORIE contains a set of synergistic structural features, none ofwhich when individually incorporated into the skeleton affords optimalactivity. Thus, with reference to FIG. 3, by examining the groupDMRIE((±)-N-(2-hydroxyethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)-1-propanaminiumbromide),DLRIE((±)-N-(2-hydroxyethyl)-N,N-dimethyl-2,3-bis(dodecyloxy)-1-propanaminiumbromide) andDDRIE((±)-N-(2-hydroxyethyl)-N,N-dimethyl-2,3-bis(decyloxy)-1-propanaminiumbromide), and comparing the groupGAP-DMRIE((+)-N-(3-aminopropyl)-N,N-dimethyl-2,3-(bis-tetradecyloxy)-1-propanaminiumbromide), GAP-DLRIE((±)-N-(3-aminopropyl)-N,N-dimethyl-2,3-(bis-dodecyloxy)-1-propanaminiumbromide), andGAP-DPRIE((±)-N-(3-aminopropyl)-N,N-dimethyl-2,3-(bis-hexadecyloxy)-1-propanaminiumbromide), it is evident that fourteen-carbon chains are more active(i.e., elicit greater levels of antibody stimulation) relative to otherchain lengths, whether the quaternary nitrogen is substituted with ahydroxyethyl moiety (former group) or with a propylamino moiety (lattergroup). By comparing DMRIE versus GAP-DMRIE (see FIG. 3), it appearsthat incorporating a propylamino group in lieu of a hydroxyethyl groupoffers no apparent advantage. Similarly, DMRIE and DMORIE are equallyactive despite the incorporation of an olefin into the fourteen-carbonchain. However, by incorporating the combination of a propylaminosubstituent and an olefin moiety, GAP-DMORIE appears to be more activethan either DMORIE or GAP-DMRIE, based on the geometric mean titer (GMT)relative to that for pDNA alone (FIG. 3). In addition, DOSPA(2,3-dioleyloxy-N-(2-(sperminecarboxamido)ethyl)-N,N-dimethyl-1-propanaminiumpentahydrochloride), which incorporates both an olefin into itseighteen-carbon alkyl chains and an amino-bearing quaternary ammoniumsubstituent, is not only less active thanDORIE((±)-N-(2-hydroxyethyl)-N,N-dimethyl-2,3-bis(syn-9-octadeceneyloxy)-1-propanaminiumbromide), which is equivalent except for quaternary ammoniumsubstitution, but dramatically decreases the level of antibody titersrelative to those seen for pDNA alone. The preferred salt of GAP-DMORIEfor use in the present invention is the bromide salt; however, allsuitable salts of GAP-DMORIE are encompassed by the term “GAP-DMORIE.”

For purposes of definition, the term “co-lipid” refers to anyhydrophobic material that can be combined with the cytofectin component,e.g., GAP-DMORIE. The co-lipid of the present invention can beamphipathic lipids and neutral lipids. Amphipathic lipids includephospholipids, e.g., phosphatidylethanolamines and phosphatidylcholines.Neutral lipids include cholesterol. In one preferred embodiment,phosphatidylethanolamines include DOPE, DMPE, and DPyPE. DOPE and DPyPEare particularly preferred; the most preferred co-lipid is DPyPE, whichcomprises two phytanoyl substituents incorporated into thediacylphosphatidylethanolamine skeleton. As illustrated by FIG. 3, thecombination of the cytofectin GAP-DMORIE with the co-lipid DPyPE resultsin a synergistic effect to further enhance the humoral immune response,as evidenced by the level of antibody titers from pDNA immunization.

According to the present invention, cytofectins and co-lipids may bemixed or combined in a number of ways to produce a variety of adjuvantcompositions of non-covalently bonded macroscopic structures, e.g.,liposomes, multilamellar vesicles, unilamellar vesicles, micelles, andsimple films. The cytofectins and co-lipids can be mixed in a variety ofmolar ratios. Preferably, the molar ratio of GAP-DMORIE and co-lipid isfrom about 9:1 to about 1:9, more preferably, the molar ratio is fromabout 4:1 to about 1:4, or from about 2:1 to about 1:2. Most preferably,the molar ratio is about 1:1.

The cytofectins and co-lipids can be dissolved in a solvent to increasehomogenity of the mixture. Suitable solvents include chloroform. Forexample, GAP-DMORIE can be mixed with one or more co-lipids inchloroform. For example, GAP-DMORIE can be mixed with one or moreco-lipids in chloroform, the mixture is subsequently evaporated undervacuum to form a dried thin layer of film on the inner surface of aglass vessel, e.g., a Rotovap round-bottomed flask. Such dried mixturecan be suspended in an aqueous solvent where the amphipathic lipidcomponent molecules self-assemble into homogenous lipid vesicles. Theselipid vesicles can subsequently be processed by any methods used in theart to have a selected mean diameter of uniform size prior to complexingwith other entities, e.g., pDNA. The sonication of a lipid solution isdescribed in Feigner et al., Proc. Natl. Acad. Sci. USA 84,7413-7417(1987) and in U.S. Pat. No. 5,264,618, the disclosure of which isincorporated herein by reference.

The adjuvant compositions of the present invention may include additivessuch as hydrophobic and amphiphilic additives. For example, the adjuvantcomposition can include sterols, fatty acids, gangliosides, glycolipids,lipopeptides, liposaccharides, neobees, niosomes, prostaglandins orsphingolipids. The amount of additives included in the adjuvant may beany including from about 0.1 mol % to about 99.9 mol %, from about 1 mol% to about 50 mol %, and from about 2 mol % to about 25 mol %, relativeto total amount of lipid. These additives can also be included in animmunogenic composition containing the adjuvant composition of thepresent invention.

The immunogenic composition of the present invention includes anadjuvant composition as described above and an immunogen. An “immunogen”is meant to encompass any antigenic or immunogenic polypeptidesincluding poly-aminoacid materials having epitopes or combinations ofepitopes, and immunogen-encoding polynucleotides. In addition, an“immunogen” is also meant to encompass any poly-saccharide materialuseful in generating immune response. As used herein, an antigenicpolypeptide or an immunogenic polypeptide is a polypeptide which, whenintroduced into a vertebrate, reacts with the immune system molecules ofthe vertebrate, i.e., is antigenic, and/or induces an immune response inthe vertebrate, i.e., is immunogenic. It is quite likely that animmunogenic polypeptide will also be antigenic, but an antigenicpolypeptide, because of its size or conformation, may not necessarily beimmunogenic. Examples of antigenic and immunogenic polypeptides include,but are not limited to, polypeptides from infectious agents such asbacteria, viruses, parasites, or fungi, allergens such as those from petdander, plants, dust, and other environmental sources, as well ascertain self polypeptides, for example, tumor-associated antigens.

Antigenic and immunogenic polypeptides of the present invention can beused to prevent or treat, i.e., cure, ameliorate, lessen the severityof, or prevent or reduce contagion of viral, bacterial, fungal, andparasitic infectious diseases, as well as to treat allergies.

In addition, antigenic and immunogenic polypeptides of the presentinvention can be used to prevent or treat, i.e., cure, ameliorate, orlessen the severity of cancer including, but not limited to, cancers oforal cavity and pharynx (i.e., tongue, mouth, pharynx), digestive system(i.e., esophagus, stomach, small intestine, colon, rectum, anus, analcanal, anorectum, liver, gallbladder, pancreas), respiratory system(i.e., larynx, lung), bones, joints, soft tissues (including heart),skin, melanoma, breast, reproductive organs (i.e., cervix, endometirum,ovary, vulva, vagina, prostate, testis, penis), urinary system (i.e.,urinary bladder, kidney, ureter, and other urinary organs), eye, brain,endocrine system (i.e., thyroid and other endocrine), lymphoma (i.e.,hodgkin's disease, non-hodgkin's lymphoma), multiple myeloma, leukemia(i.e., acute lymphocytic leukemia, chronic lymphocytic leukemia, acutemyeloid leukemia, chronic myeloid leukemia).

Examples of viral antigenic and immunogenic polypeptides include, butare not limited to, adenovirus polypeptides, alphavirus polypeptides,calicivirus polypeptides, e.g., a calicivirus capsid antigen,coronavirus polypeptides, distemper virus polypeptides, Ebola viruspolypeptides, enterovirus polypeptides, flavivirus polypeptides,hepatitis virus (AE) polypeptides, e.g., a hepatitis B core or surfaceantigen, herpesvirus polypeptides, e.g., a herpes simplex virus orvaricella zoster virus glycoprotein, immunodeficiency viruspolypeptides, e.g., the human immunodeficiency virus envelope orprotease, infectious peritonitis virus polypeptides, influenza viruspolypeptides, e.g., an influenza A hemagglutinin, neuraminidase, ornucleoprotein, leukemia virus polypeptides, Marburg virus polypeptides,orthomyxovirus polypeptides, papilloma virus polypeptides, parainfluenzavirus polypeptides, e,g., the hemagglutinin/neuraminidase, paramyxoviruspolypeptides, parvovirus polypeptides, pestivirus polypeptides, picornavirus polypeptides, e.g., a poliovirus capsid polypeptide, pox viruspolypeptides, e.g., a vaccinia virus polypeptide, rabies viruspolypeptides, e.g., a rabies virus glycoprotein G, reoviruspolypeptides, retrovirus polypeptides, and rotavirus polypeptides.

Examples of bacterial antigenic and immunogenic polypeptides include,but are not limited to, Actinomyces polypeptides, Bacillus polypeptides,Bacteroides polypeptides, Bordetella polypeptides, Bartonellapolypeptides, Borrelia polypeptides, e.g., B. burgdorferi OspA, Brucellapolypeptides, Campylobacter polypeptides, Capnocytophaga polypeptides,Chlamydia polypeptides, Clostridium polypeptides, Corynebacteriumpolypeptides, Coxiella polypeptides, Dermatophilus polypeptides,Enterococcus polypeptides, Ehrlichia polypeptides, Escherichiapolypeptides, Francisella polypeptides, Fusobacterium polypeptides,Haemobartonella polypeptides, Haemophilus polypeptides, e.g., H.influenzae type b outer membrane protein, Helicobacter polypeptides,Klebsiella polypeptides, L-form bacteria polypeptides, Leptospirapolypeptides, Listeria polypeptides, Mycobacteria polypeptides,Mycoplasma polypeptides, Neisseria polypeptides, Neorickettsiapolypeptides, Nocardia polypeptides, Pasteurella polypeptides,Peptococcus polypeptides, Peptostreptococcus polypeptides, Pneumococcuspolypeptides, Proteus polypeptides, Pseudomonas polypeptides, Rickettsiapolypeptides, Rochalimaea polypeptides, Salmonella polypeptides,Shigella polypeptides, Staphylococcus polypeptides, Streptococcuspolypeptides, e.g., S. pyogenes M proteins, Treponema polypeptides, andYersinia polypeptides, e.g., Y. pestis F1 and V antigens.

Examples of fungal immunogenic and antigenic polypeptides include, butare not limited to, Absidia polypeptides, Acremonium polypeptides,Alternaria polypeptides, Aspergillus polypeptides, Basidioboluspolypeptides, Bipolaris polypeptides, Blastomyces polypeptides, Candidapolypeptides, Coccidioides polypeptides, Conidiobolus polypeptides,Cryptococcus polypeptides, Curvalaria polypeptides, Epidermophytonpolypeptides, Exophiala polypeptides, Geotrichum polypeptides,Histoplasma polypeptides, Madurella polypeptides, Malasseziapolypeptides, Microsporum polypeptides, Moniliella polypeptides,Mortierella polypeptides, Mucor polypeptides, Paecilomyces polypeptides,Penicillium polypeptides, Phialemonium polypeptides, Phialophorapolypeptides, Prototheca polypeptides, Pseudallescheria polypeptides,Pseudomicrodochium polypeptides, Pythium polypeptides, Rhinosporidiumpolypeptides, Rhizopus polypeptides, Scolecobasidium polypeptides,Sporothrix polypeptides, Stemphylium polypeptides, Trichophytonpolypeptides, Trichosporon polypeptides, and Xylohypha polypeptides.

Examples of protozoan parasite immunogenic and antigenic polypeptidesinclude, but are not limited to, Babesia polypeptides, Balantidiumpolypeptides, Besnoitia polypeptides, Cryptosporidium polypeptides,Eimeria polypeptides, Encephalitozoon polypeptides, Entamoebapolypeptides, Giardia polypeptides, Hammondia polypeptides, Hepatozoonpolypeptides, Isospora polypeptides, Leishmania polypeptides,Microsporidia polypeptides, Neospora polypeptides, Nosema polypeptides,Pentatrichomonas polypeptides, Plasmodium polypeptides, e.g., P.falciparum circumsporozoite (PfCSP), sporozoite surface protein 2(PfSSP2), carboxyl terminus of liver state antigen 1 (PfLSA1 c-term),and exported protein 1 (PfExp-1), Pneumocystis polypeptides, Sarcocystispolypeptides, Schistosoma polypeptides, Theileria polypeptides,Toxoplasma polypeptides, and Trypanosoma polypeptides.

Examples of helminth parasite immunogenic and antigenic polypeptidesinclude, but are not limited to, Acanthocheilonema polypeptides,Aelurostrongylus polypeptides, Ancylostoma polypeptides, Angiostrongyluspolypeptides, Ascaris polypeptides, Brugia polypeptides, Bunostomumpolypeptides, Capillaria polypeptides, Chabertia polypeptides, Cooperiapolypeptides, Crenosoma polypeptides, Dictyocaulus polypeptides,Dioctophyme polypeptides, Dipetalonema polypeptides, Diphyllobothriumpolypeptides, Diplydium polypeptides, Dirofilaria polypeptides,Dracunculus polypeptides, Enterobius polypeptides, Filaroidespolypeptides, Haemonchus polypeptides, Lagochilascaris polypeptides, Loapolypeptides, Mansonella polypeptides, Muellerius polypeptides,Nanophyetus polypeptides, Necator polypeptides, Nematodiruspolypeptides, Oesophagostomum polypeptides, Onchocerca polypeptides,Opisthorchis polypeptides, Ostertagia polypeptides, Parafilariapolypeptides, Paragonimus polypeptides, Parascaris polypeptides,Physaloptera polypeptides, Protostrongylus polypeptides, Setariapolypeptides, Spirocerca polypeptides Spirometra polypeptides,Stephanofilaria polypeptides, Strongyloides polypeptides, Strongyluspolypeptides, Thelazia polypeptides, Toxascaris polypeptides, Toxocarapolypeptides, Trichinella polypeptides, Trichostrongylus polypeptides,Trichuris polypeptides, Uncinaria polypeptides, and Wuchereriapolypeptides.

Examples of ectoparasite immunogenic and antigenic polypeptides include,but are not limited to, polypeptides (including protective antigens aswell as allergens) from fleas; ticks, including hard ticks and softticks; flies, such as midges, mosquitos, sand flies, black flies, horseflies, horn flies, deer flies, tsetse flies, stable flies,myiasis-causing flies and biting gnats; ants; spiders, lice; mites; andtrue bugs, such as bed bugs and kissing bugs.

Examples of tumor-associated antigenic and immunogenic polypeptidesinclude, but are not limited to, tumor-specific immunoglobulin variableregions, GM2, Tn, sTn, Thompson-Friedenreich antigen (TF), Globo H,Le(y), MUC1, MUC2, MUC3, MUC4, MUC5AC, MUC5B, MUC7, carcinoembryonicantigens, beta chain of human chorionic gonadotropin (hCG beta),HER2/neu, PSMA, EGFRvIII, KSA, PSA, PSCA, GP100, MAGE 1, MAGE 2, TRP 1,TRP 2, tyrosinase, MART-1, PAP, CEA, BAGE, MAGE, RAGE, and relatedproteins.

Also included as polypeptides of the present invention are fragments orvariants of the foregoing polypeptides, and any combination of theforegoing polypeptides. Additional polypeptides maybe found, for examplein “Foundations in Microbiology,” Talaro, et al., eds., McGraw-HillCompanies (October 1998), Fields, et al., “Virology,” 3d ed.,Lippincott-Raven (1996), “Biochemistry and Molecular Biology ofParasites,” Man, et al., eds., Academic Press (1995), and Deacon, J.,“Modern Mycology,” Blackwell Science Inc (1997), which are incorporatedherein by reference.

The immunogen-encoding polynucleotide is intended to encompass asingular “polynucleotide” as well as plural “polynucleotides,” andrefers to an isolated molecule or construct. The immunogen-encodingpolynucleotides include nucleotide sequences, nucleic acids, nucleicacid oligomers, messenger RNA (mRNA), DNA (e.g., pDNAs, derivatives ofpDNA, linear DNA), or fragments of any of thereof. Theimmunogen-encoding polynucleotides may be provided in linear, circular,e.g., plasmid, or branched form as well as double-stranded orsingle-stranded form. The immunogen-encoding polynucleotides maycomprise a conventional phosphodiester bond or a non-conventional bond,e.g., an amide bond, such as found in peptide nucleic acids (PNA).

According to the present invention, the immunogen-encodingpolynucleotide can be part of a circular or linearized plasmidcontaining a non-infectious and non-integrating polynucleotide. Anon-infectious polynucleotide is a polynucleotide that does not infectvertebrate cells while a non-integrating polynucleotide does notintegrate into the genome of vertebrate cells. A linearized plasmid is aplasmid that was previously circular but has been linearized, forexample, by digestion with a restriction endonuclease. Theimmunogen-encoding polynucleotide may comprise a sequence that directsthe secretion of a polypeptide.

The form of immunogen-encoding polynucleotides depends in part on thedesired kinetics and duration of expression. When long-term delivery ofa protein encoded by a polynucleotide is desired, the preferred form isDNA. Alternatively, when short-term transgene protein delivery isdesired, the preferred form is mRNA, since mRNA can be rapidlytranslated into polypeptide, however RNA may be degraded more quicklythan DNA.

In one embodiment, the immunogen-encoding polynucleotide is RNA, e.g.,messenger RNA (mRNA). Methods for introducing RNA sequences intomammalian cells is described in U.S. Pat. No. 5,580,859, the disclosureof which is incorporated herein by reference. A viral alphavector, anon-infectious vector useful for administering RNA, may be used tointroduce RNA into mammalian cells. Methods for the in vivo introductionof alphaviral vectors to mammalian tissues are described inAltman-Hamamdzic, S., et al., Gene Therapy 4, 815-822 (1997), thedisclosure of which is incorporated herein by reference.

Preferably, the immunogen-encoding polynucleotide is DNA. In the case ofDNA, a promoter is preferably operably linked to the nucleotide sequenceencoding for the immunogen. The promoter can be a cell-specific promoterthat directs substantial transcription of the DNA only in predeterminedcells. Other transcription control elements, besides a promoter, can beincluded with the polynucleotide to direct cell-specific transcriptionof the DNA. An operable linkage is a linkage in which a polynucleotideencoding for an immunogenic molecule is connected to one or moreregulatory sequences in such a way as to place expression of theimmunogen under the influence or control of the regulatory sequence(s).Two DNA sequences (such as a coding sequence and a promoter regionsequence linked to the 5′ end of the coding sequence) are operablylinked if induction of promoter function results in the transcription ofmRNA encoding for the desired immunogen and if the nature of the linkagebetween the two DNA sequences does not (1) result in the introduction ofa frame-shift mutation, (2) interfere with the ability of the expressionregulatory sequences to direct the expression of the immunogen, or (3)interfere with the ability of the DNA template to be transcribed. Thus,a promoter region would be operably linked to a DNA sequence if thepromoter was capable of effecting transcription of that DNA sequence.

The immunogen-encoding polynucleotide, e.g., pDNA, mRNA, polynucleotideor nucleic acid oligomer can be solubilized in any of various buffersprior to mixing or complexing with the adjuvant components, e.g.,cytofectins and co-lipids. Suitable buffers include phosphate bufferedsaline (PBS), normal saline, Tris buffer, and sodium phosphate.Insoluble polynucleotides can be solubilized in a weak acid or weakbase, and then diluted to the desired volume with a buffer. The pH ofthe buffer may be adjusted as appropriate. In addition, apharmaceutically acceptable additive can be used to provide anappropriate osmolarity. Such additives are within the purview of oneskilled in the art.

According to the present invention, the immunogen-encodingpolynucleotides can be complexed with the adjuvant compositions of thepresent invention by any means known in the art, e.g., by mixing a pDNAsolution and a solution of cytofectin/co-lipid liposomes. In oneembodiment, the concentration of each of the constituent solutions isadjusted prior to mixing such that the desired finalpDNA/cytofectin:co-lipid ratio and the desired pDNA final concentrationwill be obtained upon mixing the two solutions. For example, if thedesired final solution is to be physiological saline (0.9%weight/volume), both pDNA and cytofectin:co-lipid liposomes are preparedin 0.9% saline and then simply mixed to produce the desired complex. Thecytofectin:co-lipid liposomes can be prepared by any means known in theart. For example, one can hydrate a thin film of GAP-DMORIE and co-lipidmixture in an appropriate volume of aqueous solvent by vortex mixing atambient temperatures for about 1 minute. Preparation of a thin film ofcytofectin and co-lipid mixture is known to a skilled artisan and can beprepared by any suitable techniques. For example, one can mix chloroformsolutions of the individual components to generate an equimolar soluteratio and subsequently aliquot a desired volume of the solutions into asuitable container where the solvent can be removed by evaporation,e.g., first with a stream of dry, inert gas such as argon and then byhigh vacuum treatment.

According to the present invention, the immunogenic composition of thepresent invention can be used to immunize a vertebrate. The term“vertebrate” is intended to encompass a singular “vertebrate” as well asplural “vertebrates”, and comprises mammalian and avian species, as wellas fish. The method for immunizing a vertebrate includes administeringto the vertebrate an immunogenic composition of the present invention inan amount sufficient to generate an immune response to the immunogen.

The immunogenic compositions of the present invention may beadministered according to any of various methods known in the art. Forexample, U.S. Pat. No. 5,676,954 reports on the injection of geneticmaterial, complex ed with cationic lipid carriers, into mice. Also, U.S.Pat. Nos. 5,589,466, 5,693,622, 5,580,859, 5,703,055, and PCTinternational patent application PCT/US94/06069 (WO 94/29469), thedisclosures of which are incorporated herein by reference, providemethods for delivering DNA-cationic lipid complexes to mammals.

Specifically, the immunogenic compositions of the present invention maybe administered to any tissue of a vertebrate, including, but notlimited to, muscle, skin, brain, lung, liver, spleen, bone marrow,thymus, heart, lymph, blood, bone, cartilage, mucosal tissue, pancreas,kidney, gall bladder, stomach, intestine, testis, ovary, uterus, vaginaltissue, rectum, nervous system, eye, gland, tongue and connectivetissue. Preferably, the compositions are administered to skeletalmuscle. The immunogenic compositions of the invention may also beadministered to a body cavity, including, but not limited to, the lung,mouth, nasal cavity, stomach, peritoneum, intestine, heart chamber,vein, artery, capillary, lymphatic, uterus, vagina, rectum, and ocularcavity.

Preferably, the immunogenic compositions of the present invention areadministered by intramuscular (i.m.) or subcutaneous (s.c.) routes.Other suitable routes of administration include transdermal, intranasal,inhalation, intratracheal, transmucosal (i e., across a mucousmembrane), intra-cavity (e.g., oral, vaginal, or rectal), intraocular,vaginal, rectal, intraperitoneal, intraintestinal and intravenous (i.v.)administration.

Any mode of administration can be used so long as the administrationresults in desired immune response. Administration means of the presentinvention include, but not limited to, needle injection, catheterinfusion, biolistic injectors, particle accelerators (i.e., “gene guns”or pneumatic “needleless” injectors—for example, Med-E-Jet (Vahlsing,H., et al., J. Immunol. Methods 171,11-22 (1994)), Pigjet (Schrijver,R., et al., Vaccine 15, 1908-1916 (1997)), Biojector (Davis, H., et al.,Vaccine 12, 1503-1509 (1994); Gramzinski, R., et al., Mol. Med. 4,109-118 (1998)), AdvantaJet, Medijector, gelfoam sponge depots, othercommercially available depot materials (e.g., hydrojels), osmotic pumps(e.g., Alza minipumps), oral or suppositorial solid (tablet or pill)pharmaceutical formulations, topical skin creams, and decanting, use ofpolynucleotide coated suture (Qin et al., Life Sciences 65, 2193-2203(1999)) or topical applications during surgery. The preferred modes ofadministration are intramuscular needle-based injection and intranasalapplication as an aqueous solution.

Determining an effective amount of an immunogenic composition dependsupon a number of factors including, for example, the chemical structureand biological activity of the substance, the age and weight of thesubject, and the route of administration. The precise amount, number ofdoses, and timing of doses can be readily determined by those skilled inthe art.

In certain embodiments, the immunogenic composition is administered as apharmaceutical composition. Such a pharmaceutical composition can beformulated according to known methods, whereby the substance to bedelivered is combined with a pharmaceutically acceptable carriervehicle. Suitable vehicles and their preparation are described, forexample, in Remington's Pharmaceutical Sciences, 16th Edition, A. Osol,ed., Mack Publishing Co., Easton, Pa. (1980), and Remington'sPharmaceutical Sciences, 19th Edition, A. R. Gennaro, ed., MackPublishing Co., Easton, Pa. (1995). The pharmaceutical composition canbe formulated as an emulsion, gel, solution, suspension, lyophilizedform, or any other form known in the art. In addition, thepharmaceutical composition can also contain pharmaceutically acceptableadditives including, for example, diluents, binders, stabilizers, andpreservatives. Administration of pharmaceutically acceptable salts ofthe polynucleotide constructs described herein is preferred. Such saltscan be prepared from pharmaceutically acceptable non-toxic basesincluding organic bases and inorganic bases. Salts derived frominorganic bases include sodium, potassium, lithium, ammonium, calcium,magnesium, and the like. Salts derived from pharmaceutically acceptableorganic non-toxic bases include salts of primary, secondary, andtertiary amines, basic amino acids, and the like.

For aqueous pharmaceutical compositions used in vivo, use of sterilepyrogen-free water is preferred. Such formulations will contain aneffective amount of the immunogenic composition together with a suitableamount of vehicle in order to prepare pharmaceutically acceptablecompositions suitable for administration to a vertebrate.

The present invention also provides kits for use in delivering apolypeptide to a vertebrate. Each kit includes a container holding 1 ngto 30 mg of an immunoge-encoding polynucleotide which operably encodesan immunogen within vertebrate cells in vivo. Furthermore, each kitincludes, in the same or in a different container, an adjuvantcomposition comprising GAP-DMORIE and a co-lipid. Any of components ofthe pharmaceutical kits can be provided in a single container or inmultiple containers. Preferably, the kit includes from about 1 ng toabout 30 mg of a immunogen-encoding polynucleotide, more preferably, thekit includes from about 100 ng to about 10 mg of a immunogen-encodingpolynucleotide.

Any suitable container or containers may be used with pharmaceuticalkits. Examples of containers include, but are not limited to, glasscontainers, plastic containers, or strips of plastic or paper.

Each of the pharmaceutical kits may further comprise an administrationmeans. Means for administration include, but are not limited to syringesand needles, catheters, biolistic injectors, particle accelerators,i.e., “gene guns,” pneumatic “needleless” injectors, gelfoam spongedepots, other commercially available depot materials, e.g., hydrojels,osmotic pumps, and decanting or topical applications during surgery.Each of the pharmaceutical kits may further comprise sutures, e.g.,coated with the immunogenic composition (Qin et al., Life Sciences(1999) 65:2193-2203).

The kit can further comprise an instruction sheet for administration ofthe composition to a vertebrate. The polynucleotide components of thepharmaceutical composition are preferably provided as a liquid solutionor they may be provided in lyophilized form as a dried powder or a cake.If the polynucleotide is provided in lyophilized form, the dried powderor cake may also include any salts, entry enhancing agents, transfectionfacilitating agents, and additives of the pharmaceutical composition indried form. Such a kit may further comprise a container with an exactamount of sterile pyrogen-free water, for precise reconstitution of thelyophilized components of the pharmaceutical composition.

The container in which the pharmaceutical composition is packaged priorto use can comprise a hermetically sealed container enclosing an amountof the lyophilized formulation or a solution containing the formulationsuitable for a pharmaceutically effective dose thereof, or multiples ofan effective dose. The pharmaceutical composition is packaged in asterile container, and the hermetically sealed container is designed topreserve sterility of the pharmaceutical formulation until use.Optionally, the container can be associated with administration meansand/or instruction for use.

The following examples are included for purposes of illustration onlyand are not intended to limit the scope of the present invention, whichis defined by the appended claims.

EXAMPLES

The following examples demonstrate the surprising finding that variousGAP-DMORIE:co-lipid complexed with an antigen-encoding pDNA can enhancesubsequent immune response compared to presently known nucleic acidimmunization methods when administered into murine or rabbit tissues.

Materials and Methods

The following materials and methods apply generally to all the examplesdisclosed herein. Specific materials and methods are disclosed in eachexample, as necessary.

Reagents

Sterile USP water and saline solutions were purchased from Baxter(Deerfield, Ill.). All other chemicals and solvents were purchasedeither from Sigma Chem. Corp. (St. Louis, Mo.) or Gallade Chemical(Escondido, Calif.). Both the1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) and1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (DPyPE) were purchasedas chloroform solutions from Avanti Polar Lipids, Inc. (Alabaster,Ala.).

Preparation of Adjuvant and Immunogenic Compositions

(±)-N-(3-aminopropyl)-N,N-dimethyl-2,3-bis(syn-9-tetradeceneyloxy)-1-propanaminiumbromide (GAP-DMORIE, also called VC 1052) was synthesized using thepublished procedure for preparing the analogue cytofectin GAP-DLRIE(Wheeler et al., Proc. Natl. Acad. Sci. 93, 11454-11459 (1996)).Specifically, substituting syn-9-tetradecenyl methane sulfonate fordodecenyl methane sulfonate in the initial bis-alkylation of3-dimethylamino-1,2-propanediol yielded the desired dialkenyl amine.Quatranization with 3-bromopropylphthalimide, followed by deprotectionof the protected primary amine with hydrazine and extractivepurification and sub-micron filtration afforded pure GAP-DMORIE asjudged by analytical thin layer chromatography. Product identity wasconfirmed using high resolution proton NMR and infrared (IR)spectroscopies.

Cytofectin:co-lipid mixtures were prepared using the rehydratedthin-film method. Briefly, dried films were prepared in 2 ml sterileglass vials by evaporating the chloroform under a stream of nitrogen,and placing the vials under vacuum overnight to remove solvent traces.Each vial contained 1.5 μmole each of a cytofectin and a co-lipid.Liposomes were prepared by adding 1 ml SWFI (sterile water forinjection, VWR, Philadelphia, Pa.) per vial followed by continuousvortexing for 5 min on the highest setting of a Genie Vortex Mixer(Fisher Scientific, Pittsburgh, Pa.). The resulting liposome solutioncontained 1.5 mM cytofectin. Formulations were prepared at finalpDNA(phosphate):cationic lipid molar ratios of 8:1, 4:1, and 2:1. Themolar concentration of pDNA phosphate is calculated by dividing the pDNAconcentration (in mg/ml) by 330, the average nucleotide molecular mass.Liposomes (in SWFI) and pDNA (in 2× vehicle) were prepared at twice thefinal concentration in the formulation. An equal volume of liposomes wasadded to pDNA using a syringe and a 26 or 28 gauge needle. Liposomeswere added in a steady stream, followed by brief, gentle vortex to mix(a few seconds on setting #4 of a Genie vortex mixer).

All cytofectin/co-lipid formulations used in this study remaineduniformly opaque for several hours after preparation at room temperaturewithout any visible aggregation. Formulations were injected 20 min-1.5hours after complexation. In a typical injection, where 5 μg of pDNA wasformulated with a cytofectin at 4:1 pDNA:cytofectin molar ratio, eachmuscle received 2.4 μg cytofectin and 3.0 μg neutral co-lipid in 50 μlof vehicle. Even the highest pDNA+cytofecin:co-lipid dose tested in themouse model (corresponding to 100 μg VR4700 plasmid+48 μg GAP-DMORIE+60μg DPyPE per mouse) did not appear to produce discomfort or result inany adverse reactions when injected into mouse muscle.

Preparation of pDNAs

The VR4700 plasmid was prepared using standard techniques known in theart. Briefly, VR1255, an optimized plasmid encoding firefly luciferase(Hartikka, J., et al., Human Gene Therapy 7, 1205-1217 (1996)), had thecoding sequence for influenza nuclear protein (NP) inserted in place ofthe luciferase coding sequence. The influenza nuclear protein sequencewas derived from a plasmid termed nCMVint-tpaPRNP (Vahlsing, L., et al.,J Immunol. Methods *174, 11-22 (1994)). More specifically, the VR4700plasmid was created via the following procedure. The VR1255 plasmid wasdigested with Acc I+Barn HI, then the ends were blunted with Klenow,thus affording the desired vector fragment. The nuclear protein codingsequence was obtained by digesting nCMVintTPAPRNP with Ace I+Eco RI, andblunting the ends with Klenow. Both the vector fragment and the insertfragment were purified, then ligated with T4 DNA ligase. The ligationproducts were transformed in E. coli to kanamycin resistance, afterwhich suitable plasmid bearing clones were identified based onrestriction digest profiles. Standard cell culture techniques were usedto expand a suitable clone, from which the plasmid was initiallyisolated and purified using well known, commercially availabletechnology (Qiagen, Valencia, Calif.).

VR1412 LacZ plasmid was constructed by subcloning a cytoplasmic-targetedβ-galactosidase gene into the VR1012 vector (Doh, S. G., et al., GeneTherapy 4(7), 268-263 (1997)). The VR1012 backbone vector contains thehuman cytomegalovirus (CMV) immediate early 1 promoter/enhancer, CMVintron A, bovine growth hormone terminator and kanamycin resistance gene(Hartikka, J., et al., Human Gene Therapy 7(10), 1205-17 (1996)).

VR5900 is a pDNA encoding hen egg lysozyme. For construction of thispDNA, gallus lysozyme cDNA was synthesized with overlappingoligonucleotides using Deep Vent DNA polymerase (NEB, Boston, Mass.).The nucleotide sequence was obtained from GENBank, accession V00428. Thesequence was humanized with the OLIGO 5.0 program and the correspondingoligonucleotides purchased from Retrogen (San Diego, Calif.). The PCRproduct was cloned into pCRII Blunt Topo (Invitrogen, Carlsbad, Calif.),sequenced in its entirety and subcloned into VR1055. VR1055 is a VicalCMV promoter/enhancer-based expression vector that is identical toVR1012 except for the use of a minimal rabbit β-globin terminator inVR1055 (Hartikka, J., et al., Human Gene Therapy 7, 1205-17 (1996)). HELexpression was confirmed by western blot with a rabbit anti-egg whitelysozyme (Biodesign, Kennebunk, Me.).

VR1904 is a pDNA encoding human factor IX. For construction, the factorIX cDNA insert from plasmid GT50 (kindly provided by Steven Josephs ofBaxter Healthcare Corp., Round Lake, Ill.) was subcloned into the VR1012vector.

VR1623 expresses a chimeric immunoglobulin with mouse variable regionsfused to human constant regions. Human kappa and gamma (IgG1) constantregions were PCR amplified from human peripheral blood lymphocytes andcloned into VR1031, a bicistronic vector derived from VR1012 byinsertion of a CITE sequence. This new construct was designated asVR1605. The variable region sequences from 38c13, a murine B-celllymphoma (Bergman and Haimovich, 1977), were amplified by PCR from theplasmid pId (Tao and Levy, 1993, kindly provided by Dr. Ronald Levy,Stanford University Medical Center, CA) and cloned into VR1605 to makeVR1623.

Bulk pDNA Preparation and Purification

Plasmid DNA was transformed into Escherichia coli DH10B or Escherichiacoli DH5α competent cells and grown in Terrific Broth (Sambrook, J., etal., in Molecular Cloning: A Laboratory Manual, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., p. A.2 (1989)) supplementedwith 50 mg/ml kanamycin in a 1 L shaker flask. Cells were harvested bycentrifugation at the end of the exponential growth phase (approximately16 hr), typically yielding 10 grams of biomass net weight per liter.Covalently closed circular pDNA was isolated by a modified lysisprocedure (Horn, N. A., et al., Human Gene Therapy 6, 565-573 (1995))followed by standard double CsCl-ethidium bromide gradientultracentrifugation with an average yield of approximately 5 mg perliter. Plasmids were ethanol precipitated and resolubilized in saline at4° C. and dialyzed against saline. Endotoxin content was determined bythe Limulus Amebocyte Lysate assay (Associates of Cape Cod, Inc.,Falmouth, Mass.). All plasmid preparations were free of detectable RNA.Endotoxin levels were less than 7.0 Endotoxin Units/mg of plasmid DNA.The spectrophotometric A₂₆₀/A₂₈₀ ratios were between 1.75 and 2.0.Plasmids were ethanol precipitated and resuspended in the injectionvehicle at 4° C. until completely dissolved. DNA was stored at −20° C.until use.

Animal Immunizations

The quadriceps muscles of restrained awake mice (female 8-12 week oldBALB/c mice from Harlan Sprague Dawley, Indianapolis, Ind.) wereinjected with pDNA in 50 μl of vehicle using a disposable insulinsyringe and a 28 gauge ½ inch needle (Becton-Dickinson, Franklin Lakes,N.J., Cat. No. 329430) fitted with a plastic collar cut from amicropipette tip. The collar length was adjusted to limit the needle tippenetration to a distance of about 2 mm into the central part of therectus femoris muscle. Injection fluids and syringes were equilibratedto room temperature and the injection of a single 50 μl volume wascarried out in 1-2 seconds.

Ketamine/xylazine anesthetized female New Zealand White rabbits (5-6months of age, approximately 3 kg) were injected in the quadricepsmuscle with 150 μg pDNA in 300 μl PBS using a 22 gauge 1 inch needle.Before injections, the injection site was shaved and cleaned withalcohol. The needle-free injection device, Biojector®2000 (Bioject Inc.,Portland, Oreg.), was tested in rabbits. The Biojector®2000 is a CO₂powered jet injection system. In a pilot experiment, it was confirmedthat the Biojector®2000 can deliver Indian ink solution through skin andinto muscle tissue.

Animal care throughout the study was in compliance with the “Guide forthe Use and Care of Laboratory Animals”, Institute of Laboratory AnimalResources, Commission on Life Sciences, National Research Council,National Academy Press, Washington, D.C., 1996 as well as with Vical'sInstitutional Animal Care and Use Committee.

Anti-NP ELISA

Ninety-six well plates (Coming Incorporated, Cat. No. 3690, Corning,N.Y.) were coated with 71-125 ng/well of influenza A/PR/8/34nucleoprotein (NP) purified from recombinant baculoviral extracts in 100μl BBS (89 mM Boric Acid+90 mM NaCl+234 mM NaOH, pH 8.3). The plateswere stored overnight at +4° C. and the wells washed twice with BBST(BBS supplemented with 0.05% Tween 20, vol/vol). The wells were thenincubated for 90 minutes with BB (BBS supplemented with 5% nonfat milk,wt/vol) and washed twice with BBST again. Two-fold serial dilutions ofmouse or rabbit serum in BB, starting at 1:20, were made in successivewells and the solutions were incubated for 2 hours at room temperature.Wells were then rinsed four times with BBST. Sera from micehyperimmunized with VR4700 NP plasmid DNA were used as a positivecontrol and pre-immune sera from mice and rabbits were used as negativecontrols.

To detect NP-specific antibodies, either alkaline phosphatase conjugatedgoat anti-mouse IgG-Fc (Jackson ImmunoResearch Laboratories, Cat. No.115-055-008, West Grove, Pa.) or goat anti-rabbit IgG-Fc (JacksonImmunoResearch Laboratories, Cat. No. 111-055-008, West Grove, Pa.)diluted 1:5000 in BBS was added at 50 μl/well and the plates wereincubated at room temperature for 2 hours. After 4 washings in BBST, 50μl of substrate (1 mg/ml p-nitrophenyl phosphate, Calbiochem Cat. No.4876 in 50 mM sodium bicarbonate buffer, pH 9.8 and 1 mM MgCl₂) wasincubated for 90 min at room temperature and absorbance readings wereperformed at 405 nm. The titer of the sera was determined by using thereciprocal of the last dilution still giving a signal two times abovebackground. Background was established using pre-immune serum diluted1:20.

Splenocyte ⁵¹Cr Release Assays

Single cell suspensions of splenocytes were pelleted and resuspended inRPMI 1640 medium containing L-glutamine and 25 mM HEPES and supplementedwith penicillin (100 U/ml), streptomycin (100 μg/ml), 55 μMβ-mercaptoethanol and 10% FBS. Unless otherwise noted, all tissueculture media and reagents were obtained from Gibco BRL LifeTechnologies (Rockville, Md.). Then, 2.5×10⁷ splenocytes were culturedfor 5 days in 25 cm² tissue culture flasks in a total of 10 ml of mediawith NP₁₄₇₋₁₅₅ peptide (H-2K^(d) TYQRTRALV) (SEQ ID NO: 1) orβ-gal₈₇₆₋₈₈₄ peptide (H-2L^(d) TPHPARIGL) (SEQ ID NO: 2) at 1 μg/ml andrecombinant murine IL-2 (Roche Molecular Bichemicals, Indianapolis,Ind.) at 0.5 U/ml.

For the CTL assay, P815 cells were labeled with 0.15 mCi Na₂ ⁵¹CrO₄ (NENLife Science Products, Boston, Mass.) in 30 μl saline at 37° C. for 35minutes. Labeled cells were pulsed with 20 μg NP peptide or β-galpeptide (H-2L^(d) TPHPARIGL) (SEQ ID NO: 2) in 1 ml RPMI 1640 media at37° C. for 40 minutes or were used unpulsed. Duplicate titrations ofsplenocytes were prepared by serially diluting the cells 1:3 in 96 wellround bottom plates (ICN Biomedicals, Aurora, Ohio). Target cells wereadded at 1×10⁴ cells/well in a final volume of 200 μl/well at thedesignated effector:target ratios (E:T). The plates were centrifuged andincubated for 4 hours at 37° C. with 5% CO₂. Counts per minute weredetermined for 100 μl of supernatant from each well. Specific lysis wascalculated as % specific lysis=[(a−b)/(c−b)]100 where a is the averagecpm released in the presence of effectors, b is the average cpm releasedfrom target cells incubated in media only and c is the cpm released fromtarget cells in the presence of 1% Triton-X 100.

EXAMPLE 1 GAP-DMORIE/Co-Lipid Enhances the Humoral Immune Response topDNA-Encoded Influenza Nucleoprotein (NP) in Mice

The present example demonstrates a quantitative comparison of theeffects of the administration of various GAP-DMORIE:co-lipid complexeswith pDNA versus pDNA alone in providing anti-NP antibody responses.

Transfection of muscles with pDNA encoding an immunogen elicits bothhumoral and cellular immune responses. To determine the extent oftransfection augmentation in an assay evaluating humoral immuneresponse, changes in anti-NP antibody levels subsequent to immunizationwith an immunogen-encoding pDNA alone, and the same pDNA complexed withvarious adjuvant compositions, were quantified. The general features ofthe immunization assay are essentially as described by Ulmer et al.(Science, 259, 1745-1749 (1993)) and uses standard ELISA technology toquantify antibody titers.

Mice were immunized using pDNA encoding influenza nuclear protein (NP),complexed with cytofectins formulated as a 1:1 (mol:mol) mixture with aco-lipid. The cytofectins were analyzed at a pDNA/cytofectin molar ratioof 4:1. Each animal in the test group (five animals per group) wasinjected with 5 μg pDNA in 50 μl physiological saline (0.9% NaClweight/volume in water) per leg in the rectus femoris muscle (10 μg pDNAtotal per animal) alone or as a cytofectin:co-lipid complex. Injectionswere performed at day “0” and at 3 weeks.

Cytofectin:co-lipid immune response enhancement was analyzed based onthe ratio of the geometric mean titer (GMT) from a cytofectin-augmentedtransfection group divided by the GMT from pDNA administration alone(see FIGS. 3 and 4). As shown in FIGS. 3 and 4, the preferred cytofectinGAP-DMORIE, when coupled with a co-lipid (especially DOPE or DPyPE),markedly enhances antibody responses to the encoded immunogen over bothpDNA alone and pDNA complexed with other cytofectin:co-lipidcombinations. Most surprisingly, the murine anti-NP antibody titers atsix weeks post-i.m. injection of VR4700 (FIG. 4) complexed withGAP-DMORIE:DPyPE resulted in a 10-fold increase in geometric meananti-NP titer.

EXAMPLE 2 GAP-DMORIE/DPyPE Enhances the Humoral Immune Response topDNA-Encoded Influenza Nucleoprotein (NP) in Mice

The purpose of the present example is to demonstrate the ability of thepreferred cytofectin:co-lipid, GAP-DMORIE/DPyPE, to enhance the humoralimmune response to pDNA-encoded NP antigen. The most preferredcytofectin:co-lipid mixture is GAP-DMORIE/DPyPE at a 1:1 molar ratio.Rather than employing the more cumbersome formal chemical nomenclatureand stipulating the specific molar ratio for the mixture, this novelformulation has been named “Vaxfectin.”

β-Galactosidase Assay

The muscle tissues were harvested, pulverized and extracted aspreviously described (Manthorpe, M., et al, Gene Quantification. Boston,Birkhauser 343-368 (1998)). The level of β-galactosidase expression inmuscle extracts was quantified using a chemiluminescent assay accordingto the manufacturer's instructions (Boehringer Mannheim, Cat. No.1758241, Indianapolis, Ind.). A standard curve, prepared in pooledextract from uninjected muscles, was included on each plate using theβ-galactosidase enzyme standard included in the kit.

Quantitation of Anti-NP Specific Antibody Secreting Cells by ELISPOTAssay

Anti-NP specific antibody secreting cells were quantified by the ELISPOTmethod using a previously described protocol (Slifka, M. K., et al, J.Virol. 69(3), 1895-1902 (1995)). Cells obtained from bone marrow (femurand tibia) were treated with 0.83% NH₄Cl to lyse red blood cells. Cellswere then resuspended in RPMI 1640 medium containing 5% fetal calf serum(Hyclone, Logan, Utah), L-glutamine, HEPES, penicillin and streptomycin(LTI, Bethesda, Md.). Nitrocellulose-bottom 96-well Multiscreenfiltration plates (Millipore Corporation, San Francisco, Calif.) werecoated with 100 μl per well of 5 μg/ml of NP antigen (influenzanucleoprotein strain A/PR/8/34) in PBS and incubated overnight at 4° C.Plates were blocked with RPMI 1640 containing 5% FBS for 2 h at roomtemperature. Blocking medium was replaced with 100 μl/well of blockingmedium containing bone marrow cell suspension obtained from miceimmunized with pDNA encoding influenza NP (with or without Vaxfectin),starting at 10⁶ cells, then diluted threefold row-wise down the plate.Control wells contained cells obtained from naive mice diluted as above(earlier controls included an irrelevant antigen). Plates were incubatedfor 5 h at 37° C. in a 7% CO₂ humidified incubator. The plates werewashed six times and incubated overnight at 4° C. with 100 μl per wellof biotinylated horse anti-mouse IgG (H+L, 1/1000 dilution, VectorLaboratories, Burlingame, Calif.) in PBS-T containing 1% FBS. Plateswere further incubated for 1 h at room temperature with 100 μl/well of 5μg/ml of horseradish peroxidase-conjugated avidin D (VectorLaboratories, Burlingame, Calif.). Antibody secreting cells weredetected by adding 100 μl per well of substrate(3-amino-9-ethylcarbazole and H₂O₂) to the plates for 3-5 minutes. Thereaction was terminated by washing profusely with tap water. Spots werecounted under a dissecting microscope. Anti-NP specific antibodysecreting cells were represented as number of spots per 10⁶ bone marrowcells.

Statistical Evaluations

All statistical comparisons were performed using the non-parametricMann-Whitney rank sum test (SigmaStat version 2.03, Jandel ScientificSoftware, San Rafael, Calif.). Differences were considered statisticallysignificant when the p value was less than 0.05.

pDNA/Vaxfectin Dose Response

To compare the effects of increasing pDNA dose, and the effect of boostinjections, mice were given bilateral i.m. injections of 1 μg, 5 μg or25 μg of naked VR4700 plasmid per muscle (thus affording a total pDNAdose of 2, 10 and 50 μg per animal, respectively) at three-weekintervals. The results are shown in FIG. 5. Higher anti-NP titers werereached when more naked pDNA was injected per muscle, and titersincreased after the first and the second boost injections. However, nofurther increase in anti-NP titers was observed with any of the pDNAdoses when a third boost injection was given at 9 weeks (data notshown), suggesting that plateau antibody titer levels had been reachedwith naked pDNA.

A second set of mice received equivalent pDNA doses formulated withVaxfectin. The results are shown in FIG. 5. Here, a 7- to 20-foldincrease in antibody titers with all three pDNA doses was observed. Thehighest average anti-NP titers per group in this experiment(204,800±56,087, n=5 mice) were measured at 9 weeks with 25 μg pDNA doseformulated with Vaxfectin. As was seen with naked pDNA injections, nofurther increase in anti-NP titers was observed with any of theVaxfectin groups when a third boost injection was given at 9 weeks (datanot shown). Thus, Vaxfectin enhanced antibody titers to levels thatcould not be reached with naked pDNA alone, either by increasing thepDNA dose or the number of injections. The most striking finding wasthat as little as 1 μg of pDNA per muscle formulated with Vaxfectinresulted in up to 5-fold higher anti-NP titer than that obtained with 25μg naked VR4700 alone.

A separate experiment was done to address whether multiple bilateralinjections are required to obtain Vaxfectin-mediated enhancement inhumoral immune response. The results are shown in Table 1. Formulating 5μg VR4700 pDNA with Vaxfectin produced a significant 6-fold increase inanti-NP titers 20 days after a single unilateral i.m. injection in mice,indicating that Vaxfectin can enhance antibody response after a singledose.

TABLE 1 Antibody titers in mouse serum after a unilateral i.m. injectionof pDNA coding for influenza nuclear protein (NP) protein^(a). Averageanti-NP titers Fold pDNA Vaxfectin Increase Day 20 710 ± 162  4,573 ±1,243^(b) 6× Day 42 5,387 ± 767   35,200 ± 6,096^(b ) 7× ^(a)Micereceived a single injection of 5 μg naked VR4700 plasmid in 50 μ1 of 150mM NaP in the right quadriceps muscle. A second group of mice received 5μg VR4700 formulated with Vaxfectin at a pDNA: cationic lipid molarratio of 4:1. On day 21, mice were given a single boost injection in thesame muscle. Total NP-specific IgG antibody titers were determined fromserum samples on day 20 and 42 (average ± S.E.M., n = 15 mice).^(b)Significantly different from naked pDNA control value (p < 0.01,Mann-Whitney rank sum test).

Formulation Optimization

Different pDNA:cationic lipid ratios were tested in the murineimmunization model to optimize Vaxfectin formulations. The results,shown in FIG. 6, indicate that injecting more pDNA-Vaxfectin complex(thus, increasing both the amount of plasmid and the amount of Vaxfectinsimultaneously) increased antibody titers in a dose dependent manner.This trend was observed for both the 2:1 and 4:1 pDNA:cationic lipidmolar ratios. When the same 5 μg pDNA dose was injected with increasingamount of Vaxfectin (thus decreasing the pDNA:cationic lipid molarratio), antibody titers again increased in a Vaxfectin dose dependentmanner (FIG. 6B). Higher pDNA doses were also examined, but injecting 50μg pDNA per limb formulated with Vaxfectin at 4:1 pDNA:cationic lipidmolar ratio did not produce any further increase in anti-NP titers,compared to 25 μg pDNA formulated with Vaxfectin at the same 4:1 ratio(data not shown).

Duration of Enhanced Humoral Response

To investigate the duration of the Vaxfectin-enhanced humoral response,NP specific antibody titers were followed for nine months after initialinjection in the murine vaccination model. The results are shown in FIG.7A. Three weeks after the boost injection given on day 21, anti-NPtiters in the Vaxfectin group were 9-fold higher than in the naked pDNAcontrol group. During the subsequent weeks, antibody titers in theVaxfectin group gradually declined but remained significantly higherthan in the controls throughout the course of the experiment. Fortyweeks after the start of the experiment, anti-NP titers in Vaxfectingroup were still 4-fold higher than in the naked pDNA group.

In a parallel experiment, another set of mice received a boost injectionon week 3, and a second identical boost at 3 months. The results areshown in FIG. 7B. The second boost injection increased antibody titersin both groups by 2- to 3-fold. However, anti-NP titers in the Vaxfectingroup appeared to remain at these elevated levels for several months,whereas the naked pDNA group yielded titers comparable to those after asingle boost at the end of the experiment. Consequently, nine monthsafter the start of the experiment, anti-NP titers in Vaxfectin groupwere 17-fold higher than in the pDNA control group.

Vaxfectin Maintains a Strong Ctl Response

It would be highly desirable that an adjuvant used in combination withpDNA vaccines to enhance Immoral immune response would not at the sametime diminish cell-mediated immunity. To evaluate this, CTL assays wereperformed after mice had been injected with various doses of pDNA withor without Vaxfectin. The results are shown in FIG. 8. Vaxfectin did nothave a significant effect on CTL response when formulated at differentpDNA and cationic lipid ratios (FIG. 8A), after a single boost (FIG. 8C)or multiple boost injections (FIGS. 8A and 8B), or when delivered in PBS(FIG. 8A) or 150 mM NaP vehicle (FIG. 8C). Injecting 25 μg of naked pDNAper muscle appeared to result in stronger CTL responses than 1 μg pDNAdose. Again, Vaxfectin did not have a significant effect on CTL responsewith either pDNA dose (FIG. 8B). Taken together, these results show thatVaxfectin could be used to enhance humoral immune response with pDNAvaccines while maintaining the strong CTL response characteristic ofpDNA immunization.

Vaxfectin Does Not Increase Muscle Transfection

To elucidate the mechanism by which Vaxfectin enhances antibodyresponses, the effect of Vaxfectin on muscle expression in vivo wasstudied. In these experiments, pDNA (VR1412) encoding β-galactosidasewas injected either alone or formulated with Vaxfectin and individualmuscles were periodically assayed for reporter gene expression. Theresults are shown in FIG. 9. One day after injections, β-galactosidaseexpression in both groups was the same, indicating that Vaxfectin didnot affect the initial transfection of muscle with pDNA. Between day 1and 7, muscle expression in the naked pDNA group increased 7-fold. Incontrast, expression in the Vaxfectin group decreased by 25% during thesame time period. Between day 7 and 21, reporter gene levels remainedthe same in the naked pDNA group, whereas β-galactosidase expression inmuscle continued to decline in the Vaxfectin group and was more than20-fold lower than in the pDNA control group at day 21. Thus, at latertime points, transgene expression in muscle was markedly reduced in theVaxfectin group, whereas antibody levels were higher. This lack ofcorrelation between muscle expression and antibody titers indicates thatVaxfectin mediated enhancement in antibody response cannot be explainedby facilitated transfection of myofibers and/or increased synthesis ofthe antigen in muscle tissue.

The mechanism by which Vaxfectin enhances the antigen-specific antibodyresponse is unclear. It is possible that Vaxfectin delivers the pDNA tomultiple cell types within muscle tissue, including antigen-presentingcells, whereas needle injection of pDNA without Vaxfectin mightprincipally transfect muscle fibers. Alternatively, the pDNA-lipidcomplex may be better able to exit the muscle and transduce distaltissue, including cells in the regional draining lymph nodes. Vaxfectincould protect the plasmid against nucleases, enabling the pDNA-lipidcomplex to reach tissues distant from the injection site. Vaxfectin mayalso induce inflammation, resulting in the damage of many transducedmuscle fibers and thereby releasing more soluble antigen soon afterinjection. A decrease in antigen production in the following days mayselect for higher affinity antigen specific B cells by limiting antigen,resulting in an increase in antibody titers.

Vaxfectin Increases the Number of Antigen-Specific Plasma Cells in BoneMarrow

Elevated anti-NP titers in Vaxfectin treated animals were maintained forseveral months after the boost injection (FIG. 7). Since long-livedplasma cells in bone marrow have been shown to be the major mechanismfor maintaining persistent antibody production after viral infection(Slifka, M. K., et al, J. Virol. 69(3), 1895-1902 (1995), Slifka, M. K.,et al, Curr. Opin. Immunol 10(3), 252-258 (1998)) the number of anti-NPantibody secreting cells from bone marrow was quantified using anELISPOT assay. The results showed that Vaxfectin produced astatistically significant 3- to 5-fold increase in the number of NPspecific plasma cells in bone marrow. Furthermore, antibody titers inindividual mice roughly correlated with the number of anti-NP antibodysecreting cells in bone marrow, both in the naked pDNA and in theVaxfectin groups (Table 2).

TABLE 2 Quantitation of anti-NP antibody secreting cells in bone marrowby ELISPOT assay PDNA Vaxfectin SFC per SFC per Anti-NP 10⁶ Anti-NP 10⁶Mouse Titer cells Mouse titer cells Exper- 1 3,200 1.8 1 51,200 17.3iment 1^(a) 2 12,800 5.0 2 102,400 11.8 3 25,600 8.3 3 102,400 29.8 451,200 13.7 4 204,800 21.2 5 204,800 34.8 Average 23,200 7.2 Average133,120 23.0^(b) Exper- 1 12,800 11.0 1 51,200 39.5 iment 2^(c) 2 12,80015.0 2 102,400 35.0 3 25,600 12.5 3 204,800 85.2 4 51,200 21.3 4 204,800132.8 Average 25,600 14.9 Average 140,800 73.1^(d) ^(a)Mice receivedbilateral intramuscular injections of 5 μg VR4700 pDNA in 50 μl PBS,either alone or formulated with Vaxfectin at 4:1 pDNA:cationic lipidmolar ratio. Identical boost injections were given at three weeks and atthree months. Mice were sacrificed four months after the start of theexperiment (one month after the second boost injection). Antibody titerswere measured from terminal bleeds and the number of anti-NP specificspot forming cells (SFC) per 10⁶ bone marrow cells were quantified.^(b)Significantly different from pDNA control value (p = 0.032,Mann-Whitney rank sum test). ^(c)Mice received bilateral intramuscularinjections of 5 μg VR4700 pDNA in 50 μl PBS, either alone or formulatedwith Vaxfectin at 4:1 pDNA:cationic lipid molar ratio. Identical boostinjections were given at three weeks and at nine months. Mice weresacrificed eleven months after the start of the experiment (two monthsafter the second boost injection). Antibody titers were measured fromterminal bleeds and anti-NP secreting cells were quantified from bonemarrow. ^(d)Significantly different from pDNA control value (p = 0.029,Mann-Whitney rank sum test).

The data from the ELISPOT assays indicate that the use of Vaxfectinincreases the number of antigen specific plasma cells in the bonemarrow. This increase in plasma cells may be due to the adjuvantproperties of the pDNA-lipid complexes. Injection of blank pDNAcomplexed with a cationic lipid into the peritoneum of murine ovariantumor bearing C3H/HeN mice induces the production of IL-6, IFN-γ, andTNF-α. (Horton, H. M., et al, J. Immunol. 163(12), 6378-6385 (1999)).These cytokines were not induced in mice treated with pDNA or lipidonly, suggesting that the pDNA-lipid complexes are immunostimulatory invivo. The immunostimulatory properties of pDNA-lipid complexes were alsoreported for experiments in which mice were injected intravenously withpDNA complexed with cationic lipid (Dow, S. W., et al, J. Immunol.163(3), 1552-1561 (1999)). As for intraperitoneal and intravenousinjection of pDNA-lipid complexes, intramuscular injection ofpDNA-Vaxfectin may also induce cytokines, including IL-6, a cytokinethat promotes the differentiation of activated B cells to plasma cells.Thus, the pDNA-Vaxfectin complexes may indirectly enhance antibodytiters by increasing the number of antibody producing B cells.

It is also possible that components of Vaxfectin might mimic naturallyoccurring mitogens that can directly stimulate polyclonal expansion of Bcells. This could enhance the specific immune response against thetransgene expressed by the muscle cells by increasing the number ofresponding B cells. Thus, increased transfection of APCs or delivery ofpDNA to the draining lymph nodes with transfection of cells in the lymphnodes, muscle damage resulting in increased availability of solubleantigen and the immunostimulatory properties of the pDNA-Vaxfectincomplexes could each contribute to the adjuvant effect of Vaxfectin.

EXAMPLE 3 Vaxfectin Enhances Antibody Titers in Rabbits

The purpose of the present example is to demonstrate the adjuvant effectof GAP-DMORIE:co-lipids (e.g., Vaxfectin) in rabbits when formulated inpolynucleotide-based vaccines.

Female New Zealand white rabbits (5-6 months old) were anesthetized,then injected in a hind leg with 300 μl of a PBS solution containingeither 150 μg of VR4700 plasmid DNA or a PBS solution containing acomplex of 150 μg VR4700 plasmid with GAP-DMORIE:DPyPE (1:1) prepared ata 4:1 mol:mol pDNA:GAP-DMORIE ratio. Each rabbit received a singleinjection using a sterile disposable, plastic insulin syringe and 22gauge 1 inch needle at day zero, plus an identical “boost” injection inthe opposite hind leg at 6 weeks. The animals were bled through an earvein prior to immunization, and at weeks 3, 6, 7, 9, and 13. Thesix-week bleed was performed the same day, but before boost injectionwas given.

Using a single unilateral i.m. injection performed with needle andsyringe, Vaxfectin produced a robust 20-fold increase in antibody titersat three weeks compared to injection of naked pDNA. The results areshown in FIG. 10. After a boost injection given at 6 weeks, anti-NPtiters in both groups increased approximately by an order of magnitude,with antibody titers in the Vaxfectin group remaining 20- to 50-foldhigher than in the naked pDNA group throughout the course of theexperiment. When rabbits were immunized with the Biojector®2000 device,Vaxfectin did not appear to enhance antibody response after a singleunilateral injection. After a boost injection was given at 6 weeks,anti-NP titers in the Biojector Vaxfectin group were up to 8-fold higherthan in the corresponding naked pDNA group.

EXAMPLE 4 Vaxfectin Enhances Antibody Production and Promotes TH1 TypeImmune Response to Various Plasmid DNA-Encoded Antigens

The purpose of the present example is to demonstrate the adjuvant effectof GAP-DMORIE:co-lipid (e.g., Vaxfectin) when formulated with variousmodel antigens, and to further characterize the immune responses to pDNAformulations containing GAP-DMORIE:co-lipid.

Immunization and Serum Collection

Restrained, awake mice received 5 μg of pDNA encoding A/PR/8/34 NP(VR4700), hen egg lysozyme (HEL, VR5900), E. coli Lac Z (β-gal, VR1412),mouse Id/human Fc (immunoglobulin variable regions from 38C13, a murinelymphoma cell line fused to a human IgG1 constant region, VR1623), orhuman factor IX (VR1904) prepared in PBS with and without Vaxfectin (50μl) and injected into the rectus femoris of 8-10 week old female mice.Mice were boosted at 3 weeks with the same dose and formulation. Micewere bled from the ophthalmic venous plexus prior to the firstinjection, 1 day prior to the boost, and at 6 weeks following the firstinjection.

IgG Antibody ELISAs

Antibody titers were determined by coating 96 well, ½ area flat wellmicrotiter plates (Corning/Costar, Inc., Corning, N.Y.) with 0.035 μginfluenza nucleoprotein (purified from recombinant baculoviralextracts), 0.25 μg hen egg lysozyme (HEL, Sigma, St. Louis, Mo.), 0.25μg E. Coli β-galactosidase (β-gal, Sigma, St. Louis, Mo.), 2.2 μg mouseId (Southern Biotech, Birmingham, Ala.), or 0.3 μg human Factor IX(Calbiochem, La Jolla, Calif.) in 50 μl BBS (89 mM Boric Acid, 90 mMNaCl pH 8.3) per well. Plates were incubated overnight at 4° C. thenwashed 4 times with BBST (BBS with 0.1% Tween 20). NP coated wells wereblocked with 100 μl of NP assay buffer (5% nonfat milk in BBS) and wellsof all other plates were blocked with 100 μl of BSA assay buffer (1%bovine serum albumin in BBS) for 1 hour at room temperature. Two-foldserial dilutions of sera in assay buffers, starting at 1:25, wereprepared and 50 μl aliquots added to each well. Following a 2-hourincubation at room temperature and 4 washes, alkaline phosphataseconjugated goat anti-mouse IgG-Fc (Jackson Immunoresearch, West Grove,Pa.) diluted 1:5000 in assay buffer was added at 50 μwell. The plateswere incubated for 2 hours at room temperature, washed 4 times and 50 μlof p-NPP substrate (1 mg/ml para-nitrophenyl phosphate, Calbiochem, LaJolla, Calif., in 50 mM sodium bicarbonate buffer, pH 9.8 and 1 mMMgCl₂) was added per well. The absorbance at 405 nm was read after 1.5hours at room temperature. The titer is the reciprocal of the lastdilution with a signal 2 times that of pre-bleed samples.

Antigen Specific IgG1 and IgG2a ELISAs

Alkaline phosphatase conjugated, mouse sub-isotype specific monoclonalantibodies were pre-titrated with standards to determine the dilution atwhich equal absorbance values were obtained for equal amounts ofstandard. For the titrations, plates were coated overnight at 4° C. with0.1 μg/50 μl/well of affinity purified goat anti-mouse kappa antisera inBBS. Plates were washed and blocked as for the NP ELISA described above.Purified mouse IgG1, κ or IgG2a, κ were serially diluted and added tothe plates at 50 μl/well. After incubating for 2 hours at roomtemperature, alkaline phophatase conjugated rat anti-mouse IgG1 andIgG2a (Pharmingen, La Jolla, Calif.) were serially diluted and added towashed plates. The assay was completed as for the NP antibody ELISA. Theassay for measurement of antigen specific sub-isotype serum titers wasas described for total IgG levels with the following modifications: thealkaline phosphatase conjugated anti-mouse IgG1 and anti-mouse IgG2awere diluted 1:1500 and 1:200 respectively.

Stimulation of CTL

Spleens were removed from euthanized mice at 11-12 weeks after the firstinjection, and 2.5×10⁷ splenocytes were cultured for 5 days in 6 wellplates in a total of 5 ml of RPMI 1640 medium (unless otherwise noted,all tissue culture reagents were obtained from Gibco BRL LifeTechnologies, Rockville, Md.) containing L-glutamine and 25 mM HEPES andsupplemented with penicillin (100 U/ml), streptomycin (100 μg/ml),5.5×10⁻⁵ M β-mercaptoethanol and 10% FBS (10% media) with eitherNP₁₄₇₋₁₅₅ peptide (H-2K^(d) TYQRTRALV) (SEQ ID NO: 1) or β-gal₈₇₆₋₈₈₄peptide (H-2L^(d) TPHPARIGL) (SEQ ID NO: 2) at 1 μg/ml and recombinantmurine IL-2 (Roche Molecular Biochemicals, Indianapolis, Ind.) at 0.5U/ml.

⁵¹Cr Release Assay

To detect antigen specific lysis, P815 cells were labeled with 0.15 mCiNa₂ ⁵¹CrO₄ (NEN Life Science Products, Boston, Mass.) and either pulsedwith 20 μg NP₁₄₇₋₁₅₅ peptide or 50 μg β-gal₈₇₆₋₈₈₄ peptide in 1 ml RPMI1640 media or were used unpulsed. Duplicate aliquots of stimulatedsplenocytes were serially diluted in 96 well round bottom plates (ICNBiomedicals, Aurora, Ohio) and target cells were added at the designatedeffector:target ratios in a final volume of 200 μl/well. The plates werecentrifuged and incubated for 4 hours at 37° C. with 5% CO₂. Afterincubation, 100 μl of supernatant from each well was analyzed. Specificlysis was calculated as % specific lysis=[(a−b)/(c−b)]100 where a is theaverage cpm released in the presence of effectors, b is the average cpmreleased from target cells incubated in media only and c is the cpmreleased from target cells in the presence of 1% Triton-X 100.

Cytokine Profiles

To determine cytokine secretion profiles of spleen cells re-stimulatedin vitro with antigen, splenocytes were plated in duplicate at 4×10⁵cells/100 μl/well in 96 well flat bottom culture plates with purified NP(purified from recombinant baculoviral extracts) or β-gal protein(Sigma, St. Louis, Mo.) at 5 μg/ml. Culture supernatants were harvestedafter 72 hours at 37° C. with 5% CO₂. Cytokines in culture supernatantswere quantified with a mouse IFN-γ ELISA kit (Pharmingen, La Jolla,Calif.) and mouse IL-4 ELISA mini-kit (Endogen, Woburn, Mass.) accordingto the manufacturers' instructions.

Statistical Analysis

Statistical analyses were performed with the 2-tailed student t-test.

Effect of Vaxfectin on Antigen Specific IgG Titers

Antigen specific antibody titers for sera collected 1 day prior to theboost at 3 weeks, and for sera collected at 6 weeks following the firstinjection are shown in FIG. 11. Immunization with pDNA/Vaxfectin had amodest effect on the three-week titers of the anti-NP and anti-Factor IXantibodies and an even greater effect on the anti-mouse id and anti-HELantibody titers. Vaxfectin had no effect on the serum titers ofanti-β-gal antibodies at 3 weeks. Three weeks following the boostimmunizations, titers for mice receiving pDNA/Vaxfectin were increasedover those receiving naked pDNA for all five of the antigens. Table 3summarizes the antigen specific IgG responses at 6 weeks for all 5 modelantigens. Vaxfectin increased the titers of pDNA induced anti-NP andanti-HEL antibodies 8 fold and 10 fold respectively over naked pDNA andincreased the titers of anti-β-gal, anti-factor IX and anti-mouse Id 3fold over naked pDNA.

TABLE 3 IgG Antibody titers at 6 Weeks* Ab Titer for Ab Titer for FoldNaked Vaxfectin Increase (average ± (average ± with Antigen std error)std error) Vaxfectin Influenza 9,821 ± 1,418 74,933 ± 8,597   8× NP HEL14,300 ± 3,798  136,720 ± 27,096  10× β-gal 27,280 ± 4,017  69,760 ±12,544  3× Mouse Id/ 972 ± 381 2,503 ± 517    3× Human Fc Human 10,240 ±2,504  30,320 ± 6,752   3× Factor IX *Mice received a bilateralinjection of 5 μg pDNA +/− Vaxfection into each rectus femoris muscle at0 and 3 weeks. Antibody titers were determined at 6 weeks (n = 20 forall groups, except for NP where n = 29 for Naked NP pDNA, n = 30 for NPp DNA/Vaxfectin, and for Mousde Idiotype where n = 19 for Naked Mouse Idp DNA).

Effect of Vaxfectin on the CTL Response

Plasmid DNA vaccination by the intramuscular route typically results instrong CTL responses to the encoded antigen (Ulmer et al., 1993; Raz etal., 1996; Donnelly et al., 1997). One possible outcome of formulatingpDNA with an adjuvant to boost antibody responses is induction of a Th2type response which could result in a weaker cell mediated immuneresponse. To determine the effect of Vaxfectin on the pDNA induced CTLresponse, spleens from mice immunized with NP or β-gal pDNA wereharvested 8-9 weeks following the boost injection. Splenocytes culturedwith NP or β-gal peptide for 5-6 days were assayed for CTL lysis of P815target cells pulsed with NP or β-gal peptide. Unpulsed P815 cells wereused to detect non-specific lysis. The antigen specific CTL effectortitration curves for % lysis of peptide pulsed target cells are shown inFIG. 12. The results for both NP and β-gal pDNA indicate thatformulation of pDNA with Vaxfectin has no significant effect on the CTLresponse at any of the effector:target ratios tested (p>0.05 at all E:Tratios).

Effect of Vaxfectin on IgG1 and IgG2a Antibody Titers

The Th1 type immune responses induced by intramuscular pDNA immunizationpromote antibody heavy chain switch in responding B cells to the IgG2asub-isotype (Raz et al., 1996). Thus production of antigen specificIgG2a is greater than antigen specific IgG1. The use of an adjuvant inpDNA vaccines could qualitatively change the immune response, resultingin greater production of either IgG1 or IgG2a. To determine the effectof Vaxfectin on the relative proportion of antigen specific serum IgG1to IgG2a when formulated with various antigen plasmid DNAs, 6 week serawere analyzed for antigen specific sub-isotype titers.

As shown in FIG. 13 a, immunizations with naked pDNA encoding differentantigens result in sub-isotype profiles that are unique to each antigen.Although the relative proportion of IgG1 and IgG2a varied for differentantigens, IgG2a was the predominant sub-isotype produced, consistentwith a Th1 type immune response. Vaxfectin formulated with all fivemodel antigen pDNAs results in an increase of both antigen specificantibody sub-isotypes (Table 4). Increases in antigen specific IgG1 andIgG2a were approximately the same magnitude for Vaxfectin formulatedpDNA for 4 of the model antigens. As compared to titers obtained withnaked pDNA, formulating pDNA with Vaxfectin increased anti-HEL IgG1titers 9-fold and IgG2a titers 11-fold. Vaxfectin increased anti-β-gal,anti-mouse Id/human Fc and anti-factor IX IgG1 titers 2 to 5-fold andIgG2a titers 2 to 4-fold over naked DNA. Vaxfectin formulated with NPpDNA increased the average anti-NP IgG1 antibody titer by 15-fold overnaked pDNA. However, the average anti-NP IgG2a antibody titer was onlyincreased 3-fold. Thus, the relative proportions of IgG1 and IgG2aelicited by immunization of pDNA/Vaxfectin remains similar to theproportions generated when naked pDNA is used to immunize mice except inthe case of NP pDNA/Vaxfectin (FIG. 13 b). In this case, there is a muchgreater increase in antibody titer of anti-NP IgG1 than anti-NP IgG2a.For all of the pDNAs formulated with Vaxfectin, titers of antigenspecific IgG2a were higher than antigen specific IgG1, suggesting a Th1type response.

TABLE 4 IgG1 and IgG2a antibody titers at 6 weeks* Influenza NP HELβ-gal Mouse Id/Human Fc Human Factor IX Isotype IgG1 IgG2a IgG1 IgG2aIgG1 IgG2a IgG1 IgG2a IgG1 IgG2a Naked 11,172 ± 213,628 ± 14,210 ±25,620 ± 8,620 ± 356,480 ± 493 ± 426 ± 6,700 ± 25,680 ± PDNA 1,82560,785 3,364 6,719 1,887 67,452 339 228 1,789 9,999 PDNA/ 171,467 ±745,813 ± 128,340 ± 280,720 ± 16,100 ± 944,640 ± 878 ± 1,615 ± 35,380 ±53,280 ± Vaxfectin 44,030 161,747 28,009 61,683 6,807 337,513 281 6389,449 11,449 Increase 15 X 3 X 9 X 11 X 2 X 3 X 2 X 4 X 5 X 2 X overNaked *Mice received a bilateral injection of 5 μg pDNA +/− Vaxfectininto each rectus femoris muscle at 0 and 3 weeks. Antibody titers weredetermined at 6 weeks (n = 20 for all groups, except for NP where n = 29for Naked NP pDNA, n = 30 for NP pDNA/Vaxfectin, and for Mouse Idiotypewhere n = 19 for Naked Mouse Id pDNA).

Effect of Vaxfectin on the Cytokine Profile

The antigen specific antibody sub-isotype analyses suggest that theresponses induced with Vaxfectin formulated pDNA, as for naked pDNA areTh1 type responses. To confirm that Vaxfectin has no effect on the Thcytokine profile in an antigen specific in vitro recall response,spleens from groups of mice immunized with NP or β-gal pDNA formulatedwith or without Vaxfectin were harvested 8-9 weeks following the boostinjection. Splenocytes were cultured and stimulated with NP or β-galprotein. Supernatants harvested from the cultured cells were assayed forIFN-γ and IL-4 production. Immunizations with NP or β-gal plasmid DNAformulated with or without Vaxfectin resulted in IFN-γ production insplenocyte cultures from all groups of immunized mice (FIG. 14). Lowlevels of IL-4 were produced in all groups of mice; however, IFN-γ wasthe predominant cytokine produced, suggesting a Th1 biased response.

In summary, the foregoing examples demonstrate the robust adjuvanteffects of a unique cationic lipid-based formulation for nucleic acidvaccines. The stimulation of the humoral response can be accomplishedwithout diminishing the strong cytolytic responses typical of nucleicacid-based vaccines. The adjuvant activity is seen in both mice andrabbits, thus implying the pharmaceutical applications in other mammals,as well as offering potential benefit in nucleic acid-based preparationof monoclonal and polyclonal antibodies. GAP-DMORIE/co-lipid (e.g.,Vaxfectin) mediated enhancement of the antibody responses was readilyobserved after a single unilateral intramuscular injection. This isimportant for the immunization of farm animals where single-shotvaccines are highly desirable since roundup of range animals isexpensive and can result in loss of production due to stress (Beard, C.W., et al, Nat. Biotechnol 16(13), 1325-1328 (1998)).

EXAMPLE 5 Human Administration

Immunogenic compositions comprising pDNA encoding hemagglutinin(HA),mixed with an adjuvant containing GAP-DMORIE formulated as a 1:1(mol:mol) mixture with DPyPE, are prepared according to the methoddescribed above. The pDNA/adjuvant molar ratio is 4:1. Three injectionsof 0.1, 0.5, 1.0, or 2.5 mg pDNA in physiological saline, as a complexwith the adjuvant, are injected into humans at 4-week intervals inalternate deltoids. Serum is removed from the humans and the HA antibodylevels are determined by serial dilution using a standard ELISA assay,as described above. Immune responses of the human subjects to the HAantibody are induced, as indicated by GMT antibody titer values.

OTHER EMBODIMENTS

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

What is claimed is:
 1. A method for increasing the immune response to anallergenic reaction in a mammal comprising administering to the mammalan immunogenic composition comprising: (a) one or moreimmunogen-encoding polynucleotides, or immunogenic polypeptides orpolysaccharides from the allergen, in an amount sufficient to increasethe immune response in the mammal to the polynucleotides, polypeptides,or polysaccharides; and (b) an adjuvant comprising a(±)-N-(3-aminopropyl)-N,N-dimethyl-2,3-bis(syn-9-tetradeceneyloxy)-1-propanaminiumsalt and one or more co-lipids, wherein the immune response to theallergenic reaction is increased in the mammal.
 2. The method of claim1, wherein the(±)-N-(3-aminopropyl)-N,N-dimethyl-2,3-bis(syn-9-tetradeceneyloxy)-1-propanaminiumsalt is(±)-N-(3-aminopropyl)-N,N-dimethyl-2,3-bis(syn-9-tetradeceneyloxy)-1-propanaminiumbromide (GAP-DMORIE).
 3. The method of claim 1, wherein the co-lipid isa phosphatidylethanolamine.
 4. The method of claim 3, wherein thephosphatidylethanolamine is1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE).
 5. The method ofclaim 3, wherein the phosphatidylethanolamine is1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (DPyPE).
 6. The methodof claim 1, wherein the molar ratio of(±)-N-(3-aminopropyl)-N,N-dimethyl-2,3-bis(syn-9-tetradeceneyloxy)-1-propanaminiumsalt to co-lipid is from about 9:1 to about 1:9.
 7. The method of claim3, wherein the phosphatidylethanolamine is1,2-dimyristoyl-glycer-3-phosphoethanolamine (DMPE).
 8. The method ofclaim 1, wherein the molar ratio of(±)-N-(3-aminopropyl)-N,N-dimethyl-2,3-bis(syn-9-tetradeceneyloxy)-1-propanaminiumsalt to co-lipid is from about 4:1 to about 1:4.
 9. The method of claim1, wherein the molar ratio of(±)-N-(3-aminopropyl)-N,N-dimethyl-2,3-bis(syn-9-tetradeceneyloxy)-1-propanaminiumsalt to co-lipid is from about 2:1 to about 1:2.
 10. The method of claim1, wherein the molar ratio of(±)-N-(3-aminopropyl)-N,N-dimethyl-2,3-bis(syn-9-tetradeceneyloxy)-1-propanaminiumsalt to co-lipid is about 1:1.
 11. The method of claim 1, wherein themammal is a human.
 12. The method of claim 1, wherein the immunogeniccomposition further comprises an excipient.
 13. The method of claim 1,wherein the administration is intravenous.
 14. The method of claim 1,wherein the administration is by a route selected from the groupconsisting of intramuscular, intratracheal, intranasal, transdermal,interdermal, subcutaneous, intraocular, vaginal, rectal,intraperitoneal, intraintestinal, and inhalation.
 15. The method ofclaim 1, wherein the polynucleotide is DNA, RNA, or a nucleic acidoligomer from the allergen.
 16. The method of claim 1, wherein thepolynucleotide is a linear or circular polynucleotide from the allergen.17. The method of claim 1, wherein the polynucleotide is all, or partof, a plasmid DNA from the allergen.